Biological assay of peptidoglycans

ABSTRACT

The present invention relates to a biological method for assaying peptidoglycans (PGN) in a sample, particularly a sample of glucose polymers. The PGN assay includes: a) treating the glucose polymer sample by sonication, heating, and/or alkalizing; b) placing the treated sample or a dilution thereof in contact with a recombinant cell expressing an exogenous TLR2 (toll-like receptor 2) and a reporter gene directly dependent on the signaling pathway associated with the TLR2. The reporter gene codes for a colored or fluorescent protein or for a protein the activity of which is measurable with or without a substrate; c) measuring the reporter gene signal; and d) determining the amount of PUN in the sample using a standard curve of the correlation between the amount of PGN and the strength of the reporter gene signal.

FIELD OF THE INVENTION

The present invention relates to assay of peptidoglycans in a sample, inparticular a sample of glucose polymers.

CONTEXT OF THE INVENTION

Aseptic inflammatory episodes are major complications observed duringtreatments using manufactured products for therapeutic purposes (forexample: peritoneal dialysis, parenteral nutrition, injection by thevenous route). Although some of these inflammatory episodes areconnected with a problem of a chemical nature (accidental presence ofchemical contaminants or incorrect dosages of certain compounds), mostcases result from the presence of contaminants of microbial originreleased during the manufacturing processes. It is now clearlyestablished that lipopolysaccharides (LPSs) and peptidoglycans (PGNs)are the main contaminants presenting a high risk of triggering suchinflammatory episodes when they are present at trace levels inmanufactured products.

The LAL (Limulus amebocyte lysate) assay is used routinely by manyquality control laboratories for detecting and assaying contaminationwith LPS. This assay is based on recognition of the endotoxins by asensing complex extracted from Limulus (horseshoe crab) hemolymph.

Other assays also based on the reactivity of extracts of invertebratehemolymph are currently proposed for detecting PGNs in products fortherapeutic use (SLP-Wako, Immunetics). However, these assays have thedisadvantage that they are not very specific, since they also react withother molecules of microbial origin, such as β-glucans. Moreover, thesemethods require purchasing special equipment for this use, which greatlyincreases the costs and therefore limits access to these assaytechniques.

Moreover, LPSs and PGNs have variable structures depending on theirbacterial origin, which is responsible for large differences ininflammatory reactivity. That is why it is in addition necessary toexpress the results of the assays in equivalent units of standardmolecules (for example, LPS of E. coli in the LAL assay).

Moreover, these molecules are most often present in the form ofmacromolecular complexes, which affects their solubility and theirinflammatory potential. For example, the PGNs are very variable in sizeand are often aggregated with other molecules of the bacterial wall,such as lipoteichoic acids and lipopeptides.

Thus, “biological” methods have been developed solely to take account ofthe inflammatory load associated with these molecules. The effectorcells of the inflammatory response possess special sensors forrecognition of molecular structures specifically produced by infectiousagents. These molecules, called PAMPs for pathogen-associated molecularpattern molecules, are essentially recognized by TLRs (Toll-likereceptors) and NLRs (Nod-like receptors), whose specificity is relatedto the molecular structure of the different classes of inflammatorymolecules. In contrast to LPS (which is a ligand recognized by type TLR4receptors), PGN is a ligand recognized by type TLR2 receptors.

In recent years, cellular assays in vitro have been developed to replacethe animal models of inflammatory response. Most of these assays arebased on the incubation of monocyte cells in the presence of thecontaminated products and on back-titration of the production ofinflammatory cytokines (TNF-α, IL-1β, IL-6, IL-8, RANTES). However,assays using primary cells isolated from blood are subject toconsiderable inter-individual variability of the donors, which may beresponsible for experimental bias.

In contrast, the monocyte cell lines give constant responses, whichexplains why they are generally preferred to primary cells. However,these lines are not completely satisfactory either. For example, thechoice of cytokines is often criticized, as most are expressedtransiently and their concentration in the culture medium does notalways reflect the real load of inflammatory molecules. Since all themonocyte cells express the majority of the TLRs/NLRs, assays based ontheir use are not selective for one type of contaminant, but will givean overall inflammatory response.

Moreover, the main problem arises from the differences in sensitivity ofthe cells with respect to the different inflammatory molecules. Thus,the PGNs, TLR2 ligands, are far less reactive than the LPSs, which makesthem difficult to detect by these approaches. In fact, the LPSs induce asignificant response for concentrations of the order of ng/mL, whereas100 times higher concentrations of PGN are necessary to obtain a similarresponse (w/w ratio).

For some years, transfected cell lines have been proposed for replacingthe above models in the biological assays for detecting and quantifyingthe reactivity of inflammatory compounds. These noninflammatory lines(for example: HEK-293) are stably transfected by a gene coding for aspecific receptor of a class of inflammatory agonists. They also containan expression vector for a reporter gene coding for an enzyme (forexample, luciferase or alkaline phosphatase), whose synthesis isdependent on activation of the inflammatory receptor. Thus, recognitionof a contaminant by the cells expressing the appropriate receptor willtrigger the synthesis of the enzyme, production of which will befollowed by transformation of its substrate into a colored orluminescent product. As this product is easily quantifiable, this methodallows rapid assay of the inflammatory response associated with a typeof contaminant.

These cellular models have many advantages: replacement of ELISA assaysof cytokines with an enzyme assay, great reproducibility of the assayson account of the stable character of the lines, targeting of certainclasses of inflammatory molecules as a function of the receptorexpressed, detection of contaminants at very low thresholds.

These cellular models may therefore replace the assays of cytokineresponse in vitro, as they make it possible to target specifically theinflammatory factors that are agonists of a given TLR or NLR, andquantify the inflammatory response associated with this agonist. Forexample, cells specifically expressing TLR2 and TLR4 have already beenused for detecting contaminants in food products (works of Clett Erridgeof the Department of Cardiovascular Sciences of Leicester—UK in BritishJournal of Nutrition, Vol. 105/issue 01/January 2011, pp 15-23).

Moreover, companies such as InvivoGen now market a wide range of cellsof the HEK-293 line (HEK-Blue™) transfected with the various TLR or NRLreceptors. These cells contain, as reporter, a gene coding for asecreted form of alkaline phosphatase (SEAP: secreted embryonic alkalinephosphatase), which allows quick and easy colorimetric assay of theresponse to the inflammatory agonists.

These HEK-Blue™ cells have already been used successfully for detectingthe presence of contaminants in concentrated solutions of glucosepolymer and their synergistic effect (WO2012/143647). As the aim statedin this application is solely to detect the contaminants that are in aform displaying inflammatory activity, the methods described in thispatent application are not suitable for measuring the total amount ofPGN contained in the sample. In fact, the soluble PGNs (MM≈120 kDa) arethose that induce an inflammatory response via the TLR2 receptor. Thus,the PGNs not having a suitable size to be inflammatory or aggregatedwith other molecules are not detected by the method described in thisapplication.

Thus, there is a constant need to develop alternative methods ofassaying total PGN in a sample, in particular a sample of glucosepolymers.

SUMMARY OF THE INVENTION

The present invention therefore relates to a biological method forassaying the peptidoglycans in a sample, in particular a sample ofglucose polymers.

In particular, the present invention relates to a method of assayingpeptidoglycans (PGNs) in a sample of glucose polymer, comprising:

-   -   a) treating the sample of glucose polymer by sonication,        heating, and/or alkalization;    -   b) bringing the treated sample or a dilution thereof into        contact with a recombinant cell expressing an exogenous TLR2        receptor (Toll-like Receptor 2) and a reporter gene under the        direct dependence of the signaling pathway associated with the        TLR2 receptor, said reporter gene coding for a colored or        fluorescent protein or for a protein whose activity can be        measured with or without substrate;    -   c) measuring the reporter gene signal; and    -   d) determining the amount of PGN in the sample using a        calibration curve of the correspondence between the amount of        PGN and the intensity of the reporter gene signal.

Preferably, treating the sample by sonication, heating, and/oralkalization makes it possible to fragment and disintegrate the PGNscontained in the sample, in particular so as to make them capable ofactivating the TLR2 receptor. In particular, the treatment of the samplemakes it possible to generate PGNs predominantly with a size of about120 kDa,

Preferably, the reporter gene is a secreted alkaline phosphatase. In apreferred embodiment, the cell is a cell of the HEK-Blue™ hTLR2 line.

Preferably, the calibration curve of the correspondence between theamount of PGN and the intensity of the reporter gene signal was preparedwith PGNs derived from a bacterium selected from Staphylococcus aureus,Micrococcus luteus, Bacillus subtilis and Alicyclobacillusacidocaldarius, preferably from Staphylococcus aureus, Micrococcusluteus, and Alicyclobacillus acidocaldarius. In particular, the methodmay comprise a preliminary step of preparation of the calibration curveusing PGNs derived from a bacterium selected from Staphylococcus aureus,Micrococcus luteus, Bacillus subtilis and Alicyclobacillusacidocaldarius, preferably from Staphylococcus aureus, Micrococcusluteus, and Alicyclobacillus acidocaldarius.

Preferably, the sample is diluted, if necessary, so as to generate asignal of the reporter gene corresponding to the linear portion of thecalibration curve.

Preferably, the sample is a sample of a solution of icodextrin.

Preferably, the calibration curve of the correspondence between theamount of PGN and the intensity of the reporter gene signal isstandardized or calibrated with an internal standard that is an agonistof TLR2, preferably a lipopeptide, in particular PAM₃Cys-Ser-(Lys)4trihydrochloride.

In an alternative embodiment, the calibration curve of thecorrespondence between the amount of PGN and the intensity of thereporter gene signal was prepared with an internal standard that is anagonist of TLR2, preferably a lipopeptide, in particularPAM₃Cys-Ser-(Lys)4 trihydrochloride. In particular, the method maycomprise a preliminary step of preparation of the calibration curveusing an internal standard that is an agonist of TLR2, preferably alipopeptide, in particular PAM₃Cys-Ser-(Lys)4 trihydrochloride.

The invention further relates to a kit for assaying peptidoglycans(PGNs) in a sample of glucose polymers, comprising:

-   -   a recombinant cell expressing an exogenous TLR2 receptor        (Toll-like Receptor 2) and a reporter gene under the direct        dependence of the signaling pathway associated with the TLR2        receptor, said reporter gene coding for a colored or fluorescent        protein or for a protein whose activity can be measured with or        without substrate; and    -   either a calibration curve of the correspondence between the        amount of PGN and the intensity of the reporter gene signal, or        a PGN standard, preferably derived from a bacterium selected        from Staphylococcus aureus, Micrococcus luteus, Bacillus        subtilis and Alicyclobacillus acidocaldarius, preferably from        Staphylococcus aureus, Micrococcus luteus, and Alicyclobacillus        acidocaldarius, or an internal standard that is an agonist of        TLR2, preferably a lipopeptide, in particular PAM₃Cys-Ser-(Lys)4        trihydrochloride;    -   optionally instructions for use and/or a solution for        pretreating the sample.

Preferably, the kit further comprises an internal standard that is anagonist of TLR2, preferably a lipopeptide, in particularPAM₃Cys-Ser-(Lys)4 trihydrochloride.

DETAILED DESCRIPTION OF THE INVENTION

The present invention therefore relates to a biological method forassaying the peptidoglycans in a sample, in particular a sample ofglucose polymers.

In particular, the present invention relates to a method of assayingpeptidoglycans (PGNs) in a sample of glucose polymer, comprising:

-   -   a) treating the sample of glucose polymer by sonication,        heating, and/or alkalization;    -   b) bringing the treated sample or a dilution thereof into        contact with a recombinant cell expressing an exogenous TLR2        receptor (Toll-like Receptor 2) and a reporter gene under the        direct dependence of the signaling pathway associated with the        TLR2 receptor, said reporter gene coding for a colored or        fluorescent protein or for a protein whose activity can be        measured with or without substrate;    -   c) measuring the reporter gene signal; and    -   d) determining the amount of PGN in the sample using a        calibration curve of the correspondence between the amount of        PGN and the intensity of the reporter gene signal.

Preferably, the glucose polymers are intended for peritoneal dialysis,enteral and parenteral nutrition and feeding of neonates. In a preferredembodiment, the glucose polymers that will be tested are icodextrin ormaltodextrins. In particular, they may be intended for preparation forperitoneal dialysis. They can be tested at one or more stages of theirpreparation, and notably at the level of the raw material, in any stepin their preparation process, and/or at the level of the end product ofthe process. They may also be tested as a sample of a solution forperitoneal dialysis.

In a first step of the method, the sample of glucose polymer is treatedby sonication, heating, and/or alkalization. The aim of this treatmentis to fragment the PGNs and/or disintegrate the PGNs contained ortrapped in aggregates, the aim being to generate PGNs capable ofinteracting with the TLR2 receptors and activating them. As statedabove, this treatment should make it possible to disintegrate the PGNscontained or trapped in aggregates and to fragment the PGNs that are toolarge, notably to generate soluble PGNs with sizes between 30 and 5000kDa, notably of about 120 kDa. However, the treatment must not affectthe capacity of the PGNs for interacting with the TLR2 receptors. It ispreferably optimized for maximum release of PGNs capable of interactingwith TLR2 and of activating the receptor and for storing a maximum ofPGNs already active on TLR2.

In a first embodiment, the treatment of the sample comprises at leastone sonication step. Optionally, sonication may take from 30 seconds to5 minutes, use a power of 20 to 40 kHz and/or comprise one or moresonication cycles, for example from 1 to 5 cycles. In a preferredembodiment, the sample will be treated by sonication for 1 minute at 35kHz in a single cycle. Optionally, the treatment by sonication may becombined with treatment by heating and/or by alkalization.

In a second embodiment, the treatment of the sample comprises at leastone alkalization step. Preferably, the alkalizing agent is NaOH, notablyat a concentration between 0.1 and 1 M. Optionally, the duration of thealkalization step may be from 5 minutes to 60 minutes. Optionally, thealkalization step may be carried out at a high temperature, notably atemperature between 20° C. and 80° C., for example at a temperature of20, 40, 60 or 80° C. Optionally, the treatment by alkalization may becombined with treatment by sonication.

In a second embodiment, the treatment of the sample comprises at leastone heating step. Optionally, the duration of the heating step may befrom 5 minutes to 60 minutes. Optionally, the heating step may becarried out at a high temperature, notably a temperature between 20° C.and 80° C., for example at a temperature of 20, 40, 60 or 80° C.Optionally, the heating treatment may be combined with treatment bysonication and/or by alkalization.

The methods of sample treatment do not comprise steps of enzymatictreatment, notably by a mutanolysin.

In a subsequent step, the sample and/or dilutions thereof is/are broughtinto contact with recombinant cells expressing the TLR2 receptor. Thecells are qualified as recombinant as they are cells that have beenmodified by the introduction of a nucleic acid coding for the TLR2receptor, preferably the human TLR2 receptor, the initial cell notexpressing TLR2.

The activity of the TLR2 receptor is detected using a reporter gene thatis under the direct dependence of the signaling pathway associated withsaid receptor. Preferably, this reporter gene codes for a colored orfluorescent protein, or for a protein whose activity can be measuredwith or without substrate. In particular, the reporter gene codes for analkaline phosphatase. Notably, the reporter gene may produce a secretedform of alkaline phosphatase (SEAP: secreted embryonic alkalinephosphatase), whose synthesis is under the direct dependence of thesignaling pathway associated with TLR2.

In a preferred embodiment, the cell line used is a HEK-10 Blue™ line(marketed by the company InvivoGen), modified by stable transfectionwith vectors coding for human TLR2: the HEK-Blue™ hTLR2 line. However,it should be noted that a person skilled in the art may also use otherlines commercially (Imgenex) or he can prepare them.

When the cell is HEK-Blue™ hTLR2, the cell is preferably used at adensity of about 50 000 cells/well for a 96-well plate.

In a particular embodiment, the sample of glucose polymers or a dilutionthereof has a concentration of glucose polymers from 5 to 50 mg/mL,preferably between 5 and 10, 20, 30 or 40 mg/mL. In the preferredembodiment, the sample of glucose polymers or a dilution thereof has aconcentration of glucose polymers of about 37.5 mg/mL.

In another particular embodiment, the sample of glucose polymers or adilution thereof has a maximum concentration of glucose polymer of 3.75%(weight/volume), preferably 3%.

For example, the samples are prepared so as to have a concentration ofglucose polymer of 3.75% (weight/volume), preferably 3%, and the samplesare submitted to the method of assay according to the present invention,assaying the sample as well as 1/10, 1/100 and 1/1000 dilutions.

Preferably, bringing the sample of glucose polymers or a dilutionthereof into contact with the cells takes about 5 to 48 h, preferablyfrom 10 to 36 h, more preferably from 16 to 24 h.

Next, the method comprises measurement of the reporter gene signal.

In a preferred embodiment using the HEK-Blue™ hTLR2 line, the signal isa measure of the activity of alkaline phosphatase. Preferably, theenzymatic reaction is carried out using a 1:3 ratio of medium to beassayed to SEAP reagent (for example 50 μL of medium and 150 μL of SEAPreagent). Moreover, a reaction time of at least 60 minutes will bepreferred.

Finally, the amount of PGN in the sample is determined using acalibration curve of the correspondence between the amount of PGN andthe intensity of the reporter gene signal.

This curve is preferably obtained with the same cells, in the sameconditions, with increasing doses of PGNs, in particular PGN standards.

The PGN standard may be any PGN of bacterial origin. For example, thePGNs may be derived from the following microorganisms: Staphylococcusaureus, Micrococcus luteus, Escherichia coli, Bacillus subtilis andAlicyclobacillus acidocaldarius. In particular, the standards used arepurified and partially digested PGNs. Such standards are availablecommercially (Invitrogen, Catalog # tlrl-pgnec or tlrl-pgnek from Ecoli; Catalog # tlrl-pgnb2 from B subtilis; Catalog # tlrl-pgnsa from Saureus) (Wako Pure Chemical, Catalog #162-18101 from M luteus).

The PGN standard is preferably calibrated using an internal standardthat is an agonist of TLR2, so as to express the results in equivalentunits of active PGN. The internal standard may be a lipopeptide,preferably synthetic, in particular PAM₃Cys-Ser-(Lys)4 trihydrochloride(Pam3(cys), PAM or Pam signifying palmitic acid) (see FIG. 5). Thus, thecalibration curve of the correspondence between the amount of PGN andthe intensity of the reporter gene signal is preferably standardized orcalibrated with an internal standard that is an agonist of TLR2,preferably a lipopeptide, in particular PAM₃Cys-Ser-(Lys)4trihydrochloride. This internal standard is preferably synthetic or witha well-defined structure/composition. The calibration or standardizationis carried out by comparing the slopes of the linear portions of eachdose-response curve and calculating a correction factor allowing thecurve obtained with the calibration standard and that of the PGNstandard to be superimposed.

For example, the calibration curve may be obtained using concentrationsof PGNs from 0.001 to 1000 ng/mL, notably from 0.01 to 100 ng/mL.

This calibration curve may be obtained either with PGNs only, or with asolution of glucose polymer to which defined quantities of PGNs havebeen added. Notably, the solution of glucose polymer used may comprise3.75% (weight/volume) of glucose polymer, preferably 3%.

This curve of the correspondence between the amount of PGN and theintensity of the reporter gene signal can also be obtained with aninternal standard that is an agonist of TLR2, preferably a lipopeptide,in particular PAM₃Cys-Ser-(Lys)4 trihydrochloride, notably with the samecells, in the same conditions, with increasing doses of TLR2 agonistinternal standard. This internal standard is preferably synthetic orwith a well-defined structure/composition. Just as for PGN, it can beobtained in the absence of or, preferably, in the presence of glucosepolymer.

Typically, the calibration curve is a classical curve of cellularresponse of the sigmoid type (FIG. 1).

-   -   part (A) corresponds to the responses obtained with low        concentrations of PGN, below those giving effective activation        of TLR2. This nonlinear zone therefore corresponds to the limit        of detection of the method. So as to include the variability of        the method, this detection threshold is estimated at three times        the value of the background noise (response obtained in the        absence of a stimulus);    -   part (B) is the most interesting as a linear response is        observed. This zone with effective response makes it possible to        determine a direct relation between the cellular response and        the PGN level. This is therefore the assay zone;    -   part (C) corresponds to saturation of the cellular response in        the presence of excessive concentrations of PGN. There is in        fact saturation of the TLR2 receptors.

The linear part of the calibration curve is considered; this partcorresponds to a zone (part B) in which the amount of PGN is directlyproportional to the reporter gene signal.

In the case of samples likely to be heavily contaminated with PGN, itwill be necessary to perform several series dilutions so as to still belocated in the zone of linearity. Otherwise, low concentrations of PGNrequire a step of concentration of the sample if we wish to increase thesensitivity of the assay.

Optionally, the method further comprises an assay with a control cellthat does not express TLR2, more generally that does not express aninnate immunity receptor. For example, the HEK-Blue™ Null2 line may beused. This is a control line, use of which is useful for verifying thatthe sample of glucose polymers does not induce production of the enzymeby an intrinsic mechanism.

The present invention also relates to a kit for assaying peptidoglycans(PGNs) in a sample of glucose polymers, said kit comprising:

-   -   a recombinant cell expressing an exogenous TLR2 receptor        (Toll-like Receptor 2) and a reporter gene under the direct        dependence of the signaling pathway associated with the TLR2        receptor, said reporter gene coding for a colored or fluorescent        protein or for a protein whose activity can be measured with or        without substrate. Notably, the cell is preferably the HEK-Blue™        hTLR2 line. As negative control, the kit may also comprise a        cell not expressing an innate immunity receptor, for example the        HEK-Blue™ Null2 line.    -   either a calibration curve of the correspondence between the        amount of PGN and the intensity of the reporter gene signal, or        a calibrated PGN standard, preferably derived from a bacterium        selected from Staphylococcus aureus, Micrococcus luteus,        Escherichia coli, and Alicyclobacillus acidocaldarius,        preferably Staphylococcus aureus, or an internal standard that        is an agonist of TLR2, preferably a lipopeptide, in particular        PAM₃Cys-Ser-(Lys)4 trihydrochloride. Optionally, the kit may        comprise both, i.e. a calibration curve as well as a calibrated        PGN standard derived from the same microorganism as that used        for preparing this calibration curve.    -   optionally instructions for use, a solution for pretreating the        sample, the reagents to be used for measuring the response of        the reporter gene, microplates, etc.

Preferably, the kit further comprises an internal standard that is anagonist of TLR2, preferably a lipopeptide, in particularPAM₃Cys-Ser-(Lys)4 trihydrochloride.

DESCRIPTION OF THE FIGURES

FIG. 1: Theoretical curve of the cellular response as a function ofincreasing concentrations of PGN.

FIG. 2: Calibration curve of the cellular response as a function of thePGN level of S. aureus obtained with the HEK-Blue™-hTLR2 cells.

FIG. 3: Response of the HEK-Blue™-hTLR2 cells as a function ofincreasing concentrations of PGN from different bacterial species.

FIG. 4: Response of the HEK-Blue™-hTLR2 cells as a function ofincreasing concentrations of PGN of S. aureus obtained from differentbatches.

FIG. 5: Structure of PAM₃Cys-Ser-(Lys)4 trihydrochloride (PAM3(cys)).

FIG. 6: Comparison of the responses induced by the PGNs of S. aureus andPAM3(cys) in the HEK-Blue™-hTLR2 cells.

FIG. 7: Response of the HEK-Blue™-hTLR2 cells as a function of thecorrected PGN concentrations.

FIG. 8: Calibration curve of the response of the HEK-Blue™-hTLR2 cellsas a function of the corrected active PGN concentrations.

EXAMPLES

The assay is based on the specific recognition of PGNs by a lineexpressing the TLR2 receptor and on the production of an enzyme activitymeasurable via activation of the signaling pathway associated with TLR2.

Cellular Material

For the experiments relating to this assay, two lines are used:

-   -   HEK-Blue™ hTLR2 line (HEK-TLR2): specific response for the TLR2        ligands, with strong reactivity for the soluble PGNs.    -   HEK-Blue™ Null2 line (HEK-Null): nonspecific response connected        with a cytotoxic effect of the sample.

The cells are cultured according to the supplier's recommendations(InvivoGen). At 75% confluence, the cells are resuspended at a densityof 0.28×10⁶ cells/mL. Before stimulation, 180 μL of the cellularsuspension is distributed in the culture wells (96-well plate), or 50000 cells/well. The cells are then stimulated for 24 h by adding 20 μLof the samples of glucose polymer at 37.5% (weight/volume) (i.e. a finaldilution of the samples at 3.75%). After 24 h of stimulation, thecellular response is measured by quantification of the enzyme activityproduced.

1—Establishment of the Dose-Response Curve

A dose-response curve was constructed by diluting different amounts ofPGN standard of S. aureus (FIG. 2) in a solution of uncontaminatedicodextrin prepared at 37.5% (weight/volume) (FIG. 2).

The result is a classical curve of cellular response of the sigmoidtype.

-   -   part (A) corresponds to the responses obtained with low        concentrations of PGN, below those giving effective activation        of TLR2. This nonlinear zone therefore corresponds to the limit        of detection of the method.    -   part (B) is the most interesting as a linear response is        observed. This zone of effective response makes it possible to        determine a direct relation between the cellular response and        the PGN level. This is therefore the assay zone.    -   part (C) corresponds to saturation of the cellular response in        the presence of excessive concentrations of PGN. There is in        fact saturation of the TLR2 receptors.

The standard curve of response of the HEK-TLR2 cells to the PGN of S.aureus has a zone of linearity for concentrations between 0.07 and 10ng/mL (i.e. between 2 and 267 ng/g of icodextrin).

2—Establishment of the Calibration Curve for Biological Assay of PGNswith an Internal Standard

The dose-response curves were constructed by diluting the PGNs ofdifferent bacterial species in a solution of uncontaminated maltodextrin(referenced P-11.11) prepared at 37.5% (weight/volume). The PGNs assayedare extracted from Staphylococcus aureus (Sigma, Cat No 77140),Micrococcus luteus (Sigma, Cat No 53243), Bacillus subtilis (InvivoGen,# tlrl-pgnb2), and Alicyclobacillus acidocaldarius (personalpreparation).

The curves obtained are classical curves of the responses observed inthe assays performed with a cellular material (bioassay) (FIG. 3). Theabsorbance values below 0.2 are evidence of PGN concentrations that aretoo low to induce a cellular response, whereas values above 2 show aplateau effect connected with saturation of the TLR2 receptors.Consequently, only the zone between these two limit values of absorbanceallows correlation of the production of SEAP with the amount of PGNpresent in the samples.

The responses observed show a large variability in the cellularreactivity associated with each type of PGN. In fact, the concentrationsgiving a response equal to 50% of the maximum response (EC50) are ˜20ng/mL for the PGNs of S. aureus and B. subtilis, 1500 ng/mL for M.luteus, and more than 2000 ng/mL for those extracted from A.acidocaldarius and E. coli K12.

However, these differences were expected, since the PGNs have differentstructures depending on their bacterial origin, which is responsible forlarge variations in inflammatory reactivity. These observationsemphasize the importance of defining an internal standard so as to beable to express the results in equivalent units of PGN.

Another factor likely to alter the response of the HEK-TLR2 cells is thesize of the PGNs, which will influence their solubility and reactivitywith respect to TLR2. Thus, the procedure for purification of thesemacromolecules may have a considerable influence on the response of thecells, since the conditions of extraction could alter the size of thePGNs, or even cause partial degradation. To test this hypothesis, theassays were reproduced with 3 separate batches of PGNs extracted from S.aureus: 2 Sigma batches (Cat No 77140: batch 1, 0001442777; batch 2,BCBH7886V) and 1 InvivoGen batch (# tlrl-pgnsa).

The results show variability of reactivity between the three batches(FIG. 4). In fact, the EC50 values are 4, 20 and 400 ng/mL respectivelyfor the three batches. These data indicate that there is a risk thatPGNs extracted from the same bacterial species might show differences inreactivity, even if the batches were obtained from the same supplier andwere extracted beforehand by the same procedure. It therefore seemsnecessary to introduce an internal standard for the calibration curve,so as to avoid errors relating to the variability of the PGNs and toexpress the results as amount of “active” PGN.

PAM₃Cys-Ser-(Lys)4 trihydrochloride (PAM3(cys); FIG. 5) is a triacylatedsynthetic lipopeptide that mimics the structure of the bacteriallipopeptides and acts as a strong agonist of TLR2. Being of homogeneousstructure, it is often used as positive control for calibrating theresponses of the cells expressing the TLR2 receptor.

The experiments were therefore reproduced replacing PGN with PAM3(cys)in our tests. As expected, the HEK-TLR2 cells show strong reactivitywith respect to this compound. Moreover, the shape of the dose-responsecurve is similar to those obtained in the presence of PGN, with EC50estimated at 10 ng/mL (FIG. 6). These results indicate that PAM3(cys)induces responses equivalent to those of the most reactive PGNs, but incontrast to the latter, it does not display structural variability.Consequently, this synthetic lipopeptide can be used for calibrating thebatches of PGN and for establishing a standardized calibration curve,which will allow the results to be formulated in amounts of “active”PGN, i.e. in amounts of PGN giving TLR2 responses identical to thoseobtained with the same amounts of PAM3 (cys).

Each batch of PGN is calibrated relative to PAM3(cys) by comparing theslopes of the linear portions of each dose-response curve, andcalculating a correction factor for superimposing the curves of the PGNson that of PAM3(cys). In the example presented in FIG. 6, the correctionfactors were estimated at 0.4, 2 and 40 for batches 1, 2 and 3,respectively. This means that 2.5 times less PGN from batch 1 isrequired for obtaining responses identical to those induced byPAM3(cys), but 2 times more PGN from batch 2 and 40 times more PGN frombatch 3. After correcting the raw quantities of PGN, it can be seen thatall the points are aligned on one and the same curve, which can besuperimposed on that obtained with PAM3(cys) (FIG. 7). Consequently,using the internal standard makes it possible to obtain correctedconcentrations for all the batches of PGN and establish a dose-responsecurve calibrated for active PGN.

By applying this method, the standard curve of response of the HEK-TLR2cells has a zone of linearity for concentrations of active PGN between0.5 and 200 ng/mL (FIG. 8), or between 13 and 5400 ng/g of glucosepolymers.

1-14. (canceled)
 15. A kit for assaying peptidoglycans (PGNs) in asample of glucose polymers comprising: a recombinant cell expressing anexogenous TLR2 receptor (Toll-like Receptor 2) and a reporter gene underthe direct dependence of the signaling pathway associated with the TLR2receptor, said reporter gene coding for a colored or fluorescent proteinor for a protein whose activity can be measured with or without asubstrate; either a calibration curve of the correspondence between theamount of PGN and the intensity of the reporter gene signal, or a PGNstandard; and optionally, instructions for use, and a solution forpretreating the sample.
 16. The kit of claim 15 wherein the PGN standardis derived from a bacterium selected from Staphylococcus aureus,Micrococcus luteus, Bacillus subtilis and Alicyclobacillusacidocaldarius.
 17. The kit of claim 15, further comprising an internalstandard that is an agonist of TLR2.
 17. The kit of claim 16, whereinthe agonist of TLR2 is a lipopeptide.
 18. The kit of claim 17, whereinsaid lipopetide is PAM3Cys-Ser-(Lys)4 trihydrochloride.
 19. The kit ofclaim 15, wherein the recombinant cell is a stably transformed HEK-293cell line.
 20. The kit of claim 15 further comprising a negativecontrol.
 21. The kit of claim 20, wherein the negative control comprisesa cell not expressing an innate immunity receptor.
 22. The kit of claim21, wherein the cell not expressing an innate immunity receptor is aHEK-null cell line.
 23. The kit of claim 15, wherein the PGN standard isa purified and partially digested PGN.
 24. The kit of claim 15 furthercomprising reagents to be used for measuring the response of thereporter gene.
 25. The kit of claim 15, wherein the reporter gene codesfor an enzyme.
 26. The kit of claim 25, wherein the enzyme is luciferaseor alkaline phosphatase.
 27. A method of using the kit of claim 15, anddetermining the presence of PGNs.